SUMMARY The pathological changes of Alzheimer?s disease (AD) develop long before the onset of symptoms, with the gradual accumulation of molecular pathology in the cerebral cortex followed by neuronal damage. While macroscopic brain changes such as hippocampal atrophy and cortical thickness are well-established as markers of symptomatic AD, the predictive value of such macroscopic imaging measures is limited prior to the onset of dementia. Diffusion MRI has emerged as a sensitive tool for probing microstructural alterations throughout the whole brain not visible on conventional MRI. The multi-scale advanced diffusion microstructural measures that are being developed for the Connectome 2.0 project for mapping differences in cortical laminar structure throughout the whole brain are timely, relevant and hold great promise for capturing the earliest microscopic gray matter abnormalities present in AD, before their overt manifestation on conventional MRI. The goal of the proposed supplement is to leverage the unique capabilities of Connectome 2.0, the next-generation human MRI scanner for imaging structural anatomy and connectivity spanning the microscopic, mesoscopic and macroscopic scales, to map alterations in neuronal microstructure and connectivity in patients with mild cognitive impairment (MCI) and AD, as well as individuals at risk of developing AD. This work will build on the hardware and acquisition advances of the Connectome 2.0 study to push the limits of diffusion MRI sensitivity for mapping gray matter microstructure and provide validated measures of cortical degeneration in MCI/AD patients and individuals at risk of developing AD. We propose to acquire advanced diffusion MRI measures on the Connectome scanner to map cortical and white matter microstructure in MCI/AD patients from the Massachusetts General Hospital Alzheimer?s Disease Research Center (MADRC) and individuals from the Human Connectome Project-Lifespan/Aging (HCP-Aging) cohort at biomarker or genetic risk for developing AD, including evidence of abnormal protein deposition on amyloid and tau PET imaging and/or presence of known genetic risk factors. Aim 1 will apply advanced diffusion MRI acquisition techniques developed for Connectome 2.0 to infer neuronal and axonal microstructure in MCI and AD patients and older adults with biomarker risk for developing AD. Aim 2 will validate diffusion MRI measures of cellular and axonal morphology in vulnerable regions of the brain in postmortem brain specimens obtained from patients with pathology-proven AD. At the end of this supplement, we anticipate the development of a validated, multi-scale advanced diffusion MRI protocol targeted for evaluating alterations in cortical and white matter microstructure and macroscopic connectivity in older adults with differing AD pathology status, with clear measures that can be eventually translated and disseminated to large-scale AD studies. Such information will inform the interpretation of diffusion imaging metrics acquired using conventional MRI scanner hardware and provide key information regarding the underlying mechanisms of disease evolution to aid in the development of future therapeutics for AD.